*Target (protein/gene name):FabI enoyl-acyl carrier protein reductase (Francisella tularensis) *NCBI Gene # or RefSeq#:3191864/NC_006570.2 *Protein ID (NP or XP #) or Wolbachia#: *Organism (including strain):Francisella tularensis Etiologic Risk Group (see link below): RG2 */Disease Information (sort of like the Intro to your Mini Research Write up): Francisella tularensis is responsible for the rare disease, Tularemia. It is most commonly found in rodents such as rabbits and hares, but it can be transmitted in humans. The disease can be spread to humans via insect bites and direct exposure to an infected animal. The highly contagious disease mainly attacks the skin, eyes, lymph nodes, and lungs. If left untreated, the disease can be fatal. There are many different types of the disease including Ulceroglandular tularemia, Oculoglandular tularemia, Oropharyngeal tularemia, and Pneumonic tularemia. The most common variant of this disease is the Ulceroglandular type. Signs and symptoms of this type include: a skin ulcer that forms at the site of infection, swollen and painful lymph glands, fever, chills, headache, and exhaustion. The common symptom among all the types of the tularemia includes ulcers at the sight of the infection. The dangerous thing about this disease is the fact that it can be used for bioterrorism. The fact that it’s highly contagious and infective means that it can pose a large threat if used as a weapon. Link to TDR Targets page (if present): N/A Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) https://www.ncbi.nlm.nih.gov/gene/3191864 Essentiality of this protein: https://www.ncbi.nlm.nih.gov/pubmed/23144254 - The Francisella tularensis FabI enoyl-acyl carrier protein reductase (FtFabI) gene is essential to bacterial viability and is expressed during infection. FtFabI is critical for fatty acid biosynthesis within the Francisella tularensis organism. The inhibition of this protein results in the loss of viability of the organism even if supplements are used. It has also ecome known that the protein is highly active during the process of infection. Without the gene, the organism will not be able to properly function normally or in times of infection. Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Monomer Complex of proteins?: Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): http://pubs.acs.org/doi/pdf/10.1021/acsinfecdis.6b00123
*EC#:EC 1.3.1.9 Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=1.3.1.9 -- Show screenshot of BRENDA enzyme mechanism schematic Figure 1. BRENDA enzyme mechanism schematic of the enoyl-[acyl-carrier-protein] reductase (NADH) Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): http://www.med.upenn.edu/ostaplab/documents/NADHLinkedAssayforATPaseMeasurements.pdf http://www.jbc.org/content/256/13/6583.full.pdf http://www.jbc.org/content/270/44/26538.full.pdf http://www.jbc.org/content/269/8/5493.long -- link to Sigma (or other company) page for assay (see Sigma links below) -- -or link (or citation) to paper that contains assay information -- links to assay reagents (substrates) pages. --- List cost and quantity of substrate reagents, supplier, and catalog #
1 mg NADH (Sigma, N0786) - $139.50 Structure (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: 2JJY -- For Homology Model option: ---- Show pairwise alignment of your BLASTP search in NCBI against the PDB ---- Query Coverage: ---- Max % Identities: ---- % Positives ---- Chain used for homology:
Current Inhibitors: Triclosan Expression Information (has it been expressed in bacterial cells): Has been expressed in E. coli http://www.jbc.org/content/270/44/26538.full.pdf Purification Method: Nickel Column Chromatography Image of protein (PyMol with features delineated and shown separately): Figure 2. PyMOL representation of the fabI enoyl-ACP reductase protein found in Francisella tularensis. The chains are colored separately and labeled (Green – A, Blue – B, Pink – C, Yellow – D). The protein is seen in its cartoon form. From 2JJY PDB file. *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
The protein is made up of 4 identical chains. Below is the sequence for a single chain:
MGSSHHHHHHSSGLVPRGSHMGFLAGKKILITGLLSNKSIAYGIAKAMHREGAELAFT
YVGQFKDREKLCAEFNPAAVLPCDVISDQEIKDLFVELGKVWDGLDAIVHSIAFAPRDQ
LEGNFIDCVTREGFSIAHDISAYSFAALAKEGRSMMKNRNASMVALTYIGAEKAMPSYN
TMGVAKASLEATVRYTALALGEDGIKVNAVSAGPIKTLAASGISNFKKMLDYNAMVSPL
KKNVDIMEVGNTVAFLCSDMATGITGEVVHVDAGYHCVSMGNVL *length of your protein in Amino Acids 280 Amino Acids x 4 = 1,120 Amino Acids Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website 29968.54 Da x 4 = 119,874.16 Da Molar Extinction coefficient of your protein at 280 nm wavelength: 17670 mol-1 cm-1 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. Figure 3. TMpred output graph for fabI enoyl-acyl carrier protein reductase found in Francisella tularensis *CDS Gene Sequence (paste as text only): ATGGGTTTTCTAGCAGGAAAAAAAATATTAATCACTGGACTTTTAAGTAATAAGTCAATTGCATATGGTA TTGCTAAAGCTATGCATAGAGAGGGAGCCGAGCTTGCTTTTACTTATGTTGGACAGTTCAAAGATAGA GTGGAAAAATTATGTGCAGAATTTAATCCAGCTGCAGTTTTGCCTTGCGATGTGATTTCTGATCAAGA GATTAAGGATTTATTTGTAGAGCTAGGTAAAGTTTGGGATGGTCTAGATGCCATAGTTCATTCTATAGC TTTTGCACCGCGTGATCAGTTAGAAGGTAACTTTATTGACTGTGTAACTCGCGAGGGTTTTAGTATCG CTCATGATATTAGTGCCTATTCTTTTGCAGCATTAGCTAAAGAAGGTCGTAGTATGATGAAAAATCGTAA TGCTTCTATGGTAGCACTTACTTATATTGGAGCAGAAAAAGCTATGCCAAGTTACAATACTATGGGAGT TGCTAAAGCATCTCTAGAAGCTACAGTTAGATATACAGCGTTAGCTTTAGGTGAGGATGGTATCAAGG TAAATGCTGTATCAGCTGGTCCTATCAAAACTCTGGCAGCTTCTGGTATATCAAACTTCAAGAAGATGC TTGATTATAATGCTATGGTTTCTCCACTTAAGAAAAATGTTGATATTATGGAAGTTGGTAATACTGTAGCG TTTTTATGTTCAGATATGGCAACTGGTATCACTGGAGAAGTTGTCCATGTTGATGCTGGATATCATTGTG TGTCTATGGTAATGTTCTTTAA *GC% Content for gene: 37.037% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
*NCBI Gene # or RefSeq#: 3191864/NC_006570.2
*Protein ID (NP or XP #) or Wolbachia#:
*Organism (including strain): Francisella tularensis
Etiologic Risk Group (see link below): RG2
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Francisella tularensis is responsible for the rare disease, Tularemia. It is most commonly found in rodents such as rabbits and hares, but it can be transmitted in humans. The disease can be spread to humans via insect bites and direct exposure to an infected animal. The highly contagious disease mainly attacks the skin, eyes, lymph nodes, and lungs. If left untreated, the disease can be fatal. There are many different types of the disease including Ulceroglandular tularemia, Oculoglandular tularemia, Oropharyngeal tularemia, and Pneumonic tularemia. The most common variant of this disease is the Ulceroglandular type. Signs and symptoms of this type include: a skin ulcer that forms at the site of infection, swollen and painful lymph glands, fever, chills, headache, and exhaustion. The common symptom among all the types of the tularemia includes ulcers at the sight of the infection. The dangerous thing about this disease is the fact that it can be used for bioterrorism. The fact that it’s highly contagious and infective means that it can pose a large threat if used as a weapon.
Link to TDR Targets page (if present): N/A
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
https://www.ncbi.nlm.nih.gov/gene/3191864
Essentiality of this protein:
https://www.ncbi.nlm.nih.gov/pubmed/23144254 - The Francisella tularensis FabI enoyl-acyl carrier protein reductase (FtFabI) gene is essential to bacterial viability and is expressed during infection. FtFabI is critical for fatty acid biosynthesis within the Francisella tularensis organism. The inhibition of this protein results in the loss of viability of the organism even if supplements are used. It has also ecome known that the protein is highly active during the process of infection. Without the gene, the organism will not be able to properly function normally or in times of infection.
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Monomer
Complex of proteins?:
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
http://pubs.acs.org/doi/pdf/10.1021/acsinfecdis.6b00123
*EC#: EC 1.3.1.9
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/enzyme.php?ecno=1.3.1.9
-- Show screenshot of BRENDA enzyme mechanism schematic
Figure 1. BRENDA enzyme mechanism schematic of the enoyl-[acyl-carrier-protein] reductase (NADH)
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
http://www.med.upenn.edu/ostaplab/documents/NADHLinkedAssayforATPaseMeasurements.pdf
http://www.jbc.org/content/256/13/6583.full.pdf
http://www.jbc.org/content/270/44/26538.full.pdf
http://www.jbc.org/content/269/8/5493.long
-- link to Sigma (or other company) page for assay (see Sigma links below)
-- -or link (or citation) to paper that contains assay information
-- links to assay reagents (substrates) pages.
--- List cost and quantity of substrate reagents, supplier, and catalog #
1 mg NADH (Sigma, N0786) - $139.50
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 2JJY
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage:
---- Max % Identities:
---- % Positives
---- Chain used for homology:
Current Inhibitors: Triclosan
Expression Information (has it been expressed in bacterial cells): Has been expressed in E. coli
http://www.jbc.org/content/270/44/26538.full.pdf
Purification Method: Nickel Column Chromatography
Image of protein (PyMol with features delineated and shown separately):
Figure 2. PyMOL representation of the fabI enoyl-ACP reductase protein found in Francisella tularensis. The chains are colored separately and labeled (Green – A, Blue – B, Pink – C, Yellow – D). The protein is seen in its cartoon form. From 2JJY PDB file.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
The protein is made up of 4 identical chains. Below is the sequence for a single chain:
MGSSHHHHHHSSGLVPRGSHMGFLAGKKILITGLLSNKSIAYGIAKAMHREGAELAFT
YVGQFKDREKLCAEFNPAAVLPCDVISDQEIKDLFVELGKVWDGLDAIVHSIAFAPRDQ
LEGNFIDCVTREGFSIAHDISAYSFAALAKEGRSMMKNRNASMVALTYIGAEKAMPSYN
TMGVAKASLEATVRYTALALGEDGIKVNAVSAGPIKTLAASGISNFKKMLDYNAMVSPL
KKNVDIMEVGNTVAFLCSDMATGITGEVVHVDAGYHCVSMGNVL
*length of your protein in Amino Acids
280 Amino Acids x 4 = 1,120 Amino Acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website
29968.54 Da x 4 = 119,874.16 Da
Molar Extinction coefficient of your protein at 280 nm wavelength: 17670 mol-1 cm-1
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Figure 3. TMpred output graph for fabI enoyl-acyl carrier protein reductase found in Francisella tularensis
*CDS Gene Sequence (paste as text only):
ATGGGTTTTCTAGCAGGAAAAAAAATATTAATCACTGGACTTTTAAGTAATAAGTCAATTGCATATGGTA
TTGCTAAAGCTATGCATAGAGAGGGAGCCGAGCTTGCTTTTACTTATGTTGGACAGTTCAAAGATAGA
GTGGAAAAATTATGTGCAGAATTTAATCCAGCTGCAGTTTTGCCTTGCGATGTGATTTCTGATCAAGA
GATTAAGGATTTATTTGTAGAGCTAGGTAAAGTTTGGGATGGTCTAGATGCCATAGTTCATTCTATAGC
TTTTGCACCGCGTGATCAGTTAGAAGGTAACTTTATTGACTGTGTAACTCGCGAGGGTTTTAGTATCG
CTCATGATATTAGTGCCTATTCTTTTGCAGCATTAGCTAAAGAAGGTCGTAGTATGATGAAAAATCGTAA
TGCTTCTATGGTAGCACTTACTTATATTGGAGCAGAAAAAGCTATGCCAAGTTACAATACTATGGGAGT
TGCTAAAGCATCTCTAGAAGCTACAGTTAGATATACAGCGTTAGCTTTAGGTGAGGATGGTATCAAGG
TAAATGCTGTATCAGCTGGTCCTATCAAAACTCTGGCAGCTTCTGGTATATCAAACTTCAAGAAGATGC
TTGATTATAATGCTATGGTTTCTCCACTTAAGAAAAATGTTGATATTATGGAAGTTGGTAATACTGTAGCG
TTTTTATGTTCAGATATGGCAACTGGTATCACTGGAGAAGTTGTCCATGTTGATGCTGGATATCATTGTG
TGTCTATGGTAATGTTCTTTAA
*GC% Content for gene: 37.037%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.